Mesoscale sample preparation device and systems for determination and processing of analytes

ABSTRACT

A mesoscale sample preparation device capable of providing microvolume test samples, separated into a cell-enriched fraction and a fraction of reduced cell content, for performing various analyses, such as binding assays, determinations involving polynucleotide amplification and the like. Analytical systems including such devices are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. application Ser. No. 08/338,369, filed Nov.14, 1994, now U.S. Pat. No. 5,726,026, the last mentioned applicationbeing a continuation-in-part of the following applications: U.S. Ser.No. 07/877,702, filed May. 1, 1992, now abandoned; U.S. Ser. No.08/196,021, filed Feb. 14, 1994, now U.S. Pat. No. 5,635,358, which is adivisional of U.S. Ser. No. 07/877,536, filed May 1, 1992, now U.S. Pat.No. 5,304,487; U.S. Ser. No. 08/250,100, filed May, 26, 1994, nowabandoned, which is a continuation of U.S. Ser. No. 07/877,701, filedMay 1, 1992, now abandoned; and U.S. Ser. No. 08/308,199, filed Sep. 19,1994, now U.S. Pat. No. 5,498,392, which is a continuation of U.S. Ser.No. 07/877,662, filed May 1, 1992 now abandoned. The entire disclosuresof the aforementioned patents and patent applications are incorporatedby reference herein.

BACKGROUND OF THE INVENTION

This invention relates to sample preparation devices having smalldimensions for facilitating the efficient preparation of microvolumetest samples, e.g., of whole blood, for the determination and/orprocessing of analytes present therein. The present invention alsorelates to test systems including such devices, together with devices ofsimilar dimensions which are designed, for example, to perform variousassay protocols as well as analyses involving amplification ofpre-selected polynucleotides, such as polymerase chain reaction (PCR).

In recent decades the art has developed a large number of protocols,test kits, and devices for conducting analyses on biological samples forvarious diagnostic and monitoring purposes. Immunoassays, immunometricassays, agglutination assays, analyses involving polynucleotideamplification reactions, various ligand-receptor interactions, anddifferential migration of species in a complex sample all have been usedto determine the presence or quantity of various biological molecules orcontaminants, or the presence of particular cell types.

Recently, small, disposable devices have been developed for handlingbiological samples and for conducting certain clinical tests. Shoji etal. reported the use of a miniature blood gas analyzer fabricated on asilicon wafer. Shoji et al., Sensors and Actuators, 15: 101-107 (1988).Sato et al. reported a cell fusion technique using micromechanicalsilicon devices. Sato et al., Sensors and Actuators, A21-A23: 948-953(1990). Ciba Corning Diagnostics Corp. (USA) has manufactured amicroprocessor-controlled laser photometer for detecting blood clotting.

Micromachining technology originated in the microelectronics industry.Angell et al., Scientific American, 248: 44-55 (1983). Micromachiningtechnology has enabled the manufacture of microengineered devices havingstructural elements with minute dimensions, ranging from tens of microns(the dimensions of biological cells) to nanometers (the dimensions ofsome biological macromolecules). Most experiments reported to dateinvolving such small structures have involved studies of micromechanics,i.e., mechanical motion and flow properties. The potential capability ofsuch devices has not been exploited fully in the life sciences.

Brunette (Exper. Cell Res., 167: 203-217 (1986) and 164: 11-26 (1986))studied the behavior of fibroblasts and epithelial cells in grooves insilicon, titanium-coated polymers and the like. McCartney et al. (CancerRes., 41: 3046-3051 (1981)) examined the behavior of tumor cells ingrooved plastic substrates. LaCelle (Blood Cells, 12: 179-189 (1986))studied leukocyte and erythrocyte flow in microcapillaries to gaininsight into micro-circulation. Hung and Weissman reported a study offluid dynamics in micromachined channels, but did not produce dataassociated with an analytical device. Hung et al., Med. and Biol.Engineering, 9: 237-245 (1971); and Weissman et al., Am. Inst. Chem.Eng. J., 17: 25-30 (1971). Columbus et al. utilized a sandwich composedof two orthogonally orientated v-grooved embossed sheets in the controlof capillary flow of biological fluids to discrete ion-selectiveelectrodes in an experimental multi-channel test device. Columbus etal., Clin. Chem., 33: 1531-1537 (1987). Masuda et al. and Washizu et al.have reported the use of a fluid flow chamber for the manipulation ofcells (e.g., cell fusion). Masuda et al., Proceedings IEEE/IAS Meeting,pp. 1549-1553 (1987); and Washizu et al., Proceedings IEEE/IAS Meeting,pp. 1735-1740 (1988). The art has not fully explored the potential ofusing microengineered devices for the determination of analytes in fluidsamples, particularly in the area of biological analyses.

Biological analyses utilizing polynucleotide amplification techniquesare well known (See e.g., Maniatis et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1989, pp.14.1-14.35). One such technique is PCR amplification, which can beperformed on a DNA template using a thermostable DNA polymerase, e.g.,Taq DNA polymerase (Chien et al., J. Bacteriol., 127: 1550 (1976)),nucleoside triphosphates, and two oligonucleotides with differentsequences, complementary to sequences that lie on opposite strands ofthe template DNA and which flank the segment of DNA that is to beamplified ("primers"). The reaction components are cycled between ahigher temperature (e.g., 94° C.) for dehybridizing double strandedtemplate DNA, followed by lower temperatures (e.g., 65° C.) forannealing and polymerization. A repeated reaction cycle betweendehybridization, annealing and polymerization temperatures providesapproximately exponential amplification of the template DNA. Machinesfor performing automated PCR chain reactions using a thermal cycler areavailable (Perkin Elmer Corp.) PCR amplification has been applied to thediagnosis of genetic disorders (Engelke et al., Proc. Natl. Acad. Sci.,85: 544 (1988), the detection of nucleic acid sequences of pathogenicorganisms in clinical samples (Ou et al., Science, 239: 295 (1988)), thegenetic identification of forensic samples, e.g., sperm (Li et al.,Nature, 335: 414 (1988)), the analysis of mutations in activatedoncogenes (Farr et al., Proc. Natl. Acad. Sci., 85: 1629 (1988)) and inmany aspects of molecular cloning (Oste, BioTechniques, 6: 162 (1988)).PCR assays can be used in a wide range of applications such as thegeneration of specific sequences of cloned double-stranded DNA for useas probes, the generation of probes specific for uncloned genes byselective amplification of particular segments of cDNA, the generationof libraries of cDNA from small amounts of mRNA, the generation of largeamounts of DNA for sequencing, and the analysis of mutations. There is aneed for convenient, rapid systems for performing polynucleotideamplification, which may be used clinically in a wide range of potentialapplications in clinical tests such as tests for paternity, and forgenetic and infectious diseases.

Current analytical technicues utilized for the determination ofmicroorganisms are rarely automated, usually require incubation in asuitable medium to increase the number of organisms, and generallyemploy visual and/or chemical methods to identify the strain orsub-species of interest. The inherent delay in such methods frequentlynecessitates medical intervention prior to definitive identification ofthe nature of an infection. In industrial, public health or clinicalenvironments, such delays may have unfortunate consequences. There is aneed for convenient systems for the rapid detection of microorganisms.

It is an object of the present invention to provide sample preparationdevices for use with related analytical devices which enable rapid andefficient analysis of sample fluids, based on very small volumes, anddetermination of substances present therein at very low concentrations.Another object is to provide easily mass produced, disposable, small(e.g., less than 1 cc in volume) devices having microfabricatedstructural elements capable of facilitating rapid, automated analyses ofpreselected molecular or cellular analytes, including intracellularmolecules, such as DNA, in a range of biological and other applications.It is a further object of the invention to provide a variety of suchdevices that individually can be used to implement a range of rapidclinical tests, e.g., tests for viral or bacterial infection, geneticscreening, sperm motility, blood parameters, contaminants in food,water, or body fluids, and the like.

SUMMARY OF THE INVENTION

The present invention provides a microfabricated sample preparationdevice which conveniently provides microvolume fractions of test samplecomprising particulate components, e.g., cells, for various biologicaland other analyses. The invention further provides analytical systemswhich include the microfabricated sample preparation device of theinvention together with a microfabricated analyte detection device,e.g., an immunoassay device, and/or a microfabricated device forcarrying out polynucleotide amplification.

The sample preparation device of the present invention comprises asample flow path having a sample inlet and an outlet in fluidcommunication and a separator disposed between the inlet and the outlet.The separator has an upstream-facing portion defining a separation zonein the flow path in which particulate components present in the samplefluid are collected. The device preferably comprises a flow channel influid communication with the separation is zone which affords dischargeof collected particulate components from the separation zone. The flowchannel has an inlet section for directing a carrier fluid into theseparation zone and over the upstream-facing portion of the separatorand a discharge section for directing the carrier fluid from over theupstream-facing portion of the separator and out of the separation zone.At least one of the flow path and the flow channel sections has at leastone mesoscale dimension, as characterized below.

According to one embodiment of the invention, the flow path has at leastone mesoscale dimension and the separator comprises a region ofrestricted flow in the flow path, which is formed by at least onepassageway having at least one mesoscale dimension that is smaller thanthe least mesoscale dimension of the flow path and sufficiently small toseparate particulate components from the sample fluid.

The sample preparation device of the invention can be made using knownmicrofabrication techniques, with the flow path and the flow channelbeing formed in a surface of a solid substrate. In a preferredembodiment, the surface of the substrate in which the structuralelements are formed is enclosed by a cover, such as a transparent glassor plastic cover, adhered to such surface.

The mesoscale sample preparation device of the present invention isspecially adapted for use in conjunction with the mesoscale detectiondevices which are the subject of co-pending U.S. Ser. No. 07/877,702,now abandoned, and/or the mesoscale polynucleotide amplification deviceswhich are the subject of co-pending U.S. Ser. No. 08/038,199 now U.S.Pat. No. 5,493,392. The full disclosures of the '702 and '199applications are incorporated by reference in the present application,as if set forth herein in full, as previously noted.

The mesoscale devices described above can be used in variouscombinations to function as an analytical system, as will be describedin further detail below. In one embodiment, the devices may be utilizedfor analyses of a cell-containing test sample. The test sample fractionsprovided by the sample preparation device of the present invention maybe analyzed serially or essentially simultaneously.

The mesoscale detection devices, which enable the determination ofvarious analytes of interest, comprise a solid substrate microfabricatedto define a sample inlet port and a mesoscale flow system which includesan analyte detection region in fluid communication with the inlet portand, optionally, a flow channel interconnecting the inlet port and theanalyte detection region. At least one of the analyte detection regionand the sample flow channel, when present, has at least one mesoscaledimension. The analyte detection region is provided with a reagent whichinteracts with the analyte of interest, resulting in a detectableproduct which is determinative of the analyte. In one embodiment, thereagent is a binding substance, optionally immobilized in the detectionregion, either on a stationary or mobile support, for specificallybinding the analyte. Also included is a detector for detecting theaforementioned product, which allows determination of the analyte in thetest sample.

The mesoscale polynucleotide amplification device comprises a solidsubstrate that is microfabricated to define a sample inlet port and amesoscale flow system, which includes a polynucleotide amplificationregion in fluid communication with the inlet port of the devices, and,optionally, a flow channel interconnecting the inlet port and thepolynucleotide amplification region. At least one of the polynucleotideamplification region and the sample flow channel, when the latter ispresent, has at least one mesoscale dimension. Lysing means is alsoprovided in a sample flow channel upstream of the polynucleotideamplification region for lysing cell components of a biological testsample. Such devices may be utilized to implement PCR, in which case thepolynucleotide amplification region contains appropriate reagents andmeans is provided for thermally cycling the reagents, such that, in eachcycle, the temperature is controlled to dehybridize double strandedpolynucleotides, anneal the primers to single stranded polynucleotide,and synthesize amplified polynucleotide between the primers.

The individual analytical devices described herein are within the scopeof the present invention, whether or not they are used in conjunctionwith the sample preparation device of the invention.

The devices described above will normally be used with an appliance thatfunctions as a holder for the devices and which mates one or more portson the devices with one or more flow lines in the appliance. A testsample, such as whole blood, containing an analyte of interest may beapplied to the inlet of the sample preparation device after which animpellent, such as a pump, which may be incorporated in the appliance orin the device itself, is employed to cause the sample to flow along theflow path and through the separation zone. Test sample which is free ofparticulate components is transferred from the sample preparation deviceto the analyte detection device, the outlet of the former being in fluidcommunication with the inlet port of the latter. Particulate components,such as blood cells or other formed bodies, remaining in the separationzone can be discharged from the separation zone, and transferred to thepolynucleotide amplification device via the discharge section of theflow channel of the sample preparation device, which is in fluidcommunication with the inlet port of the polynucleotide amplificationdevice. Alternatively, the test sample may be injected into the samplepreparation device, or the sample may enter the mesoscale samplepreparation device through the inlet by capillary action. optionally,depending on the analytical protocol being carried out in the devicesdescribed above, the appliance may also be designed to inject into thedevices reagents, such as labelled binding substances, polynucleotideamplification reagents, buffers, or any other reagent required to carryout the desired analysis.

The device and systems of the invention may be used to implement avariety of automated, sensitive and rapid clinical tests including theanalysis of cells or molecules or for monitoring reactions or cellgrowth. Essentially any test involving determination of the presence orconcentration of a molecular or ionic analyte, the presence of aparticular cell type or the presence of a gene or recombinant DNAsequence in a cell can be implemented to advantage using the device andanalytical systems of the present invention. These mesoscale devices canprovide a rapid chemical test for the detection of pathogenic bacteriaor viruses. The devices can also provide a rapid test for the presenceor concentration of blood constituents, such as hormones. Additionaluseful applications include, but are not limited to, a range of otherbiological assays, such as blood type testing.

The device and systems of the invention may be readily sterilized priorto use. Tests performed using the device and systems of the inventionmay be completed rapidly, and at the conclusion of the test the devicescan be discarded, which beneficially prevents contamination betweensamples, entombs potentially hazardous material, produces onlymicrovolumes of waste fluid for disposal and enables inexpensiveanalyses.

Additional advantages and features of the present invention are setforth in, and will be apparent to those skilled in the art from thedetailed description of the invention presented below, considered inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a diagrammatic representation of asample preparation device of the invention, as seen through atransparent cover.

FIGS. 2 and 3 show fragmentary plan views of different embodiments of amicrofabricated restricted flow (filter-type) separator within the flowpath through a portion of a sample preparation device, the separatorhaving a series of passageways restricting flow of the test samplethrough the flow path.

FIG. 4 is a schematic illustration, in cross-section, of a samplepreparation device of the invention combined with an appliance whichserves to hold the device and to regulate fluid flow through the device.

FIG. 5 is a plan view of a diagrammatic representation of the samedevice shown in FIG. 1, the respective outlets of which are in fluidcommunication with first and second microfabricated analyticalstructures which are designed to perform separate analyses on the samplefractions provided by the sample preparation device.

FIGS. 6A and 6B are schematic illustrations, in cross-section, of ELsample preparation device of the invention with the outlet of the flowpath from the separation zone in fluid communication with the sampleinlet of an analytical device for implementing various assay protocols.Both devices are shown in combination with an appliance which serves tohold the devices, regulate fluid flow through the devices, and, in theembodiment shown in FIG. 6A, detect pressure differentials atpreselected locations along the course of fluid flow through thedevices. FIG. 6A shows the devices abutting end-to-end; and FIG. 6Bshows a stacked arrangement of the devices.

FIG. 7 is a schematic illustration, in cross-section, of a samplepreparation device of the invention with the outlet of the carrier fluidflow channel in fluid communication with the sample inlet of ananalytical device for performing polynucleotide amplification. Bothdevices are shown in combination with an appliance which serves to holdthe devices, regulate fluid flow through the devices and detect pressuredifferentials at preselected locations along the course of fluid flowthrough the devices.

FIGS. 8A and 8B show, in plan view, diagrammatic illustrations of twoanalytical devices intended for use with the sample preparation deviceof the invention. The device of FIG. 8A has two mesoscale flow systems,each one including inlet ports interconnected by a flow channel to asingle chamber for analyte capture and, optionally, detection. FIG. 8Bshows a similar design for performing enzyme immunoassays and havingdual capture chambers. An analyte of interest, such as a protein, may becaptured in the first chamber, e.g., by a suitable immunocapturereagent, labelled with an antibody-enzyme conjugate and exposed to achromogenic substrate. The enzyme converts the substrate to achromophore which is captured, e.g., by a suitable immunocapturereagent, in the second chamber which concentrates the chromophore andreduces background signal. The second chamber may optionally be used fordetection of the chromophore, as well.

FIG. 9 is a plan view of a diagrammatic representation of amicrofabricated analytical device intended for use with the samplepreparation device of the invention. The analytical device includes aset of tortuous channels which enable the timed addition and mixing ofreagents, wash liquids and the like used in conducting various assayprotocols. As seen in FIG. 9A, a single chamber is provided for captureand detection of the analyte of interest; FIG. 9B shows an exploded viewof a part of an alternative embodiment of the device having an analytecapture chamber and a separate analyte detection chamber; FIG. 9C showsan exploded view of part of another embodiment of the device including abranched flow path region which permits analyte detection based on flowrestriction in the branched region.

FIG. 10A is a plan view of a diagrammatic representation of anotherembodiment of an analytical device for carrying out various assayprotocols on microvolume samples, which may be used together with thesample preparation device of the present invention;

FIG. 10B is an exploded fragmentary plan view of a part of the firstflow passage through which sample fluid flows upon its introduction intothe sample inlet port of the device shown in FIG. 10A;

FIG. 10C is a fragmentary transverse cross-section of the first flowpassage taken along the line 10C--10C in FIG. 10B, showing theside-by-side v-shape channels which constitute the first flow passage;

FIG. 10D is a fragmentary longitudinal cross-section of the first flowpassage taken along the line 10D--10D in FIG. 10C, showing certainstructural features of the barrier separating the v-shaped channels;

FIG. 11A is a plan view of a diagrammatic representation of ananalytical device intended for use with the sample preparation device ofthe invention, the analytical device having a series of mesoscalechambers suitable for implementing a variety of procedures includingcell sorting, cell lysing and polynucleotide amplification, e.g., PCR;FIG. 11B is a plan view of a diagrammatic illustration of an alternativedesign for a mesoscale PCR analytical device.

FIGS. 12A and 12B are fragmentary plan views of additional embodimentsof microfabricated, restricted flow separators disposed in the flow pathof a sample preparation device of the invention.

FIGS. 12C and 12D are fragmentary longitudinal sectional views of otheradditional embodiments of microfabricated restricted flow separatorsdisposed in the flow path of the sample preparation device of theinvention.

Like reference characters designate like parts in the drawing figures inwhich they appear.

DETAILED DESCRIPTION OF THE INVENTION

The sample preparation device of the invention comprises a solidsubstrate, preferably in the form of a chip having dimensions on theorder of less than one to a few millimeters thick and approximately 0.1to 5.0 centimeters square. The substrate is microfabricated to form asample flow path having an inlet and an outlet as well as a separatordisposed intermediate to the inlet and outlet. The upstream-facingportion of the separator defines a separation zone in the flow path inwhich particulate components of the test sample are collected. Thedevice may also include a flow channel in fluid communication with theseparation zone which functions to discharge collected particulatecomponents from the separation zone. The flow channel has an inletsection for directing a carrier fluid into the separation zone and overthe upstream-facing portion of the separator and a discharge section fordirecting the carrier fluid, in which the particulate components areentrained, out of the separation zone. At least one of theaforementioned flow path and flow channel sections have at least onemesoscale dimension.

If the particulate components of the sample are not to be analyzed, theycan remain in the separation zone, in which case the flow channel isessentially nonfunctional and thus may be eliminated from the device.

As used herein, the term "mesoscale" refers to flow passages or channelsand other structural elements, e.g. reaction and/or detection chambers,at least one of which has at least one cross-sectional dimension on theorder of 0.1 μm to 1000 μm and more preferably 0.2 μm to 500 μm. Thepreferred depth of the flow passages and chambers is on the order of0.1-100 μm and more preferably 2-50 μm. The preferred flow passage widthis on the order of 2-200 μm and more preferably 3-100 μm. The preferredchamber width is on the order of 0.05-5 mm and more preferably 50-500μm. The width of the passageway(s) in the separator is typically on theorder of less than 50 μm which is sufficiently small to separateparticulate matter from most biological samples and other test samplesof interest. The separator passageways will normally have a depth ofabout 0.1 to about 100 μm. The length of the separator passageways willtypically be within the range of about 0.1 μm to about 5 mm.

The flow passages and other structures, when viewed in cross-section,may be triangular, ellipsoidal, square, rectangular, circular or anyother shape at least one cross-sectional dimension of which, transverseto the path of flow of sample fluid through or into a given structure,is mesoscale.

The mesoscale devices of the invention facilitate sample preparation ina broad range of biological analyses and, together with the analyticaldevices described herein, enable the rapid determination ofmicroquantities of both molecular and cellular analytes in various testsamples. At the conclusion of the analysis, the devices typically arediscarded.

Mesoscale devices having at least one flow passage or other structuralelement with at least one mesoscale dimension can be designed andfabricated in large quantities from a solid substrate material usingvarious micromachining methods known to those skilled in the art. Suchmethods include film deposition processes, such as spin coating andchemical vapor deposition, laser machining or photolithographictechniques, e.g. UV or X-ray processes, etching methods which may beperformed by either wet chemical processes or plasma processes, LIGAprocessing or plastic molding. See, for example, Manz et al., Trends inAnalytical Chemistry 10:144-149 (1991).

The sample preparation device of the invention may be convenientlyconstructed by forming the flow passages and separator in the surface ofa suitable substrate and then mounting a cover over such surface. Thesolid substrate and/or cover may comprise a material such as silicon,polysilicon, silica glass, thermocouple materials, gallium arsenide,polyimide, silicon nitride and silicon dioxide. The cover and/orsubstrate may also comprise a plastic material, such as acrylic,polycarbonate, polystyrene, polyethylene or other resin materials.optionally, the cover and/or substrate may comprise a transparentmaterial, e.g., a relatively thin, anodically bonded layer of glass orultrasonically welded plastic sheet material. Alternatively, twosubstrates of like material can be sandwiched together, or a suitablesubstrate material may be sandwiched between two transparent coverlayers.

A diagrammatic representation of one embodiment the mesoscale samplepreparation device of the invention is shown in FIG. 1. The device 10 ismicrofabricated in a suitable substrate 11, thereby forming a sampleflow path 12a and 12b having sample inlet port 14 and outlet port 16. Afilter-type separator 18 is interposed in the flow path between inlet 14and outlet 16. The upstream-facing portion 20 of the separator defines aseparation zone 22 for collecting particulate components of the testsample. The device also includes a flow channel 24a and 24b in fluidcommunication with separation zone 22 for delivering a carrier fluid to,and discharging collected particulate matter from the separation zone.Flow channel 24a, 24b has an inlet section 26 for directing carrierfluid, e.g., isotonic buffer, from a source (not shown) over theupstream-facing portion 20 of separator 18. Discharge section 28 conveysthe carrier fluid from over the upstream-facing surface of the filterelement and out of separation zone 22.

Separator 18 which is microfabricated in sample flow path 12a and 12b ofthe sample preparation device serves to remove particulate matter fromthe test sample passed through the device prior to analysis. In oneembodiment, shown in FIGS. 2 and 3, the separator comprises a series ofmesoscale passageways of reduced dimension in comparison with flow path12a, 12b. In operation, separator 18 functions as a filter, accumulatingparticulate matter on its upstream surface 18a, while the filtrateexiting passageways 19 continues along flow path 12b. The filterpassageways 19 are microfabricated with depths and widths on the orderof about 5 μm to about 50 μm, whereas flow paths 12a, 12b have maximumdepths and widths on the order of approximately 1000 μm. The filterelement is preferably microfabricated in the substrate of the device soas to form at least one, and preferably several, generally upstandingprojections of the substrate material disposed in the flow path, whichserve to restrict the flow of sample fluid through the separation zone.

Protuberances p may be provided on the exterior of the upstream-facingportion of separator 18, as depicted in FIG. 2, as an aid in preventingplugging of passageways 19 by particulate matter in the sample fluid.Also, a sump (not shown) may be provided adjacent the upstream-facingportion of separator 18 for collecting insoluble debris removed from thesample fluid.

Separator 18 preferably is an essentially stationary structurepermanently positioned between sample inlet 14 and outlet 16 of the flowpath, as can be seen in FIG. 1. Alternatively, however, the separatormay be transiently disposed in the flow path. For example, a mass ofmagnetic particles may be retained in relatively fixed position in flowpath 12a, 12b by means of an applied magnetic field to effect filtrationof particulate matter from the test sample. The fluid portion of thesample passes through the void spaces between the particles as thefiltrate. At the appropriate time, the applied magnetic field is removedand the magnetic particles may be transferred from the flow path,together with any particular matter from the test sample accumulatedthereon, for analysis or disposal, as desired.

Separator 18 may, if desired, comprise a reagent that facilitatesremoval of particles or formed bodies from the test sample. In the caseof a biological sample comprising a mixed cell population, for example,a binding substance that releasably binds to a specific target cell typewithin the mixed population may be adsorbed or otherwise affixed to theseparator to effect removal and selective retention of the target celltype. Cells which are not retained can be conveyed from the separationzone for disposal. The retained cells are subsequently caused to bereleased for analysis.

The sample preparation device of the invention can be used incombination with an appliance, such as appliance 30, shown in schematiccross-section in FIG. 4, for delivering fluids to, discharging fluidsfrom, and transferring fluids between the different devices constitutingthe analytical systems of the invention. Appliance 30, which has anesting site 32 for holding the device 10, and for registering ports,e.g. port 14 on the device, with a flow line 33 in the appliance. Theappliance may include an impellent, such as pump 34 shown in FIG. 4, forconveying the sample through the flow passages of the device. After abiological fluid sample suspected to contain a particular analyte ofinterest is applied to the inlet port 35 of the appliance, pump 34 isactuated to convey the sample into port 14 of device 10 and then throughflow path 12a, 12b. Although pump 34 is shown as an element of appliance30, it may, if desired be incorporated into device 10 according to knownmicrofabrication techniques. Economic considerations, however, favorplacement of the pump in appliance 30. Alternatively, depending on thenature of the analyses to be performed, a sample may be injected intothe device, or the sample may enter the flow passages of the devicethrough the inlet port by capillary action. In another embodiment, theappliance may be disposed over the sample preparation chip, and may beprovided with a flow line communicating with the inlet port in thedevice, e.g., in the absence of a cover over the device, to allow asample to be injected into the device. The microfabricated structures ofthe devices may be filled to a hydraulically full volume and theappliance may be utilized to direct the flow of fluid through thestructures, e.g., by means of valves located in the device or in theappliance. the incorporation of valves in a microfabricated silicon chipcan be accomplished according to techniques known in the art.

The outlet 36 of appliance 30 may be interconnected to the inlet of asimilar appliance holding an analytical device of the type describedherein, whereby the sample prepared in device 10 is transferred to theanalytical device for testing.

The analytical devices also may be utilized in combination with anappliance for viewing the contents of the mesoscale flow passages andother structures in the devices. For example, the appliance may comprisea microscope (not shown) for viewing the contents of the mesoscalestructure(s) in the device. Transparent cover 29, as shown in FIG. 1,serves as a window which facilitates dynamic viewing of the contents ofthe device.

FIG. 5 shows a diagrammatic representation of the combination of thesample preparation device of FIG. 1 and analytical device 110 designedto carry out various binding assay protocols, and also polynucleotideamplification. To this end the device 110 is provided with an assaystructure 112 and a polynucleotide amplification/assay structure 122. Inthe embodiment illustrated in FIG. 5, the outlet of flow path 12a, 12bis in fluid communication with the inlet port 114 of assay structure 112of the device; and the discharge section 28 of channel 24a, 24b is influid communication with the inlet port 124 of polynucleotideamplification/assay structure 122. Reagents used in performing the assayor other test or analysis may be introduced through reagent inlet ports116 or 126, respectively. A reaction region 117 is typically provided inassay structure 112 in which a suitable reagent interacts with theanalyte to yield a detectable product which is determinative of theanalyte. That is to say, the product produced is one which providesdefinite information as to the nature or quantity of the analyte. Theproduct may be detected in the form in which it is produced in reactionregion 117, or it may be subject to further reaction to enhance itsdetection. A separate reaction/detection region 118 may be provided forthis purpose.

A solution containing analyte-specific binding substances may beintroduced into reaction region 117 via an inlet port (not shown) influid communication with the reaction region. Protein binding substancesintroduced in aqueous solution may be retained in a mesoscale structurein lyophilized form. Alternatively, binding substances may beimmobilized in a mesoscale chamber of the analytical devices after itsmanufacture by, for example, physical adsorption or chemical attachmentto the surface of the chamber or to a mobile, solid phase support, suchas magnetic or non-magnetic polymer particles disposed in the chamber.

In carrying out polynucleotide amplification using device 110, cells ofinterest transferred from discharge section 28 of the sample preparationdevice 10 are subject to lysis either by a lysing agent or by a lysingstructure as described in the above-mentioned U.S. Pat. No. 5,304,487.The target polynucleotide released from the cells undergoesamplification in amplification region 127 and the amplifiedpolynucleotide may be detected in detection region 128. One or more ofthe apertures 116, 119, 126 and 129 may be open to the atmosphere tovent the system(s). The operation of the binding assay structure 112 andthe polynucleotide amplification/assay structure 122 will be furtherexplained with reference to other embodiments of such devices describedbelow.

Although assay structure 112 and polynucleotide amplification/assaystructure 122 are fashioned on a common substrate as a single device, asshown in FIG. 5, the structures may be fabricated on separate substratesand function as distinct analytical devices or chips, as will appearbelow.

When the sample preparation device and analytical devices describedabove are used together to function as a analytical system, asillustrated in FIG. 5, for example, the system is advantageouslycombined with an appliance of the type depicted in FIGS. 6A, 6B and 7.Like the appliance of FIG. 4, previously described, appliance 50 in FIG.6A serves to deliver fluid to, discharge fluid from, and transfer fluidbetween the respective devices. Appliance 50 has a nesting site 52 forholding sample preparation device 10 and analytical device 112 and forregistering ports in the devices with flow lines in the appliance.Specifically, flow line 54a is in registry with inlet port 14 of thesample preparation device, flow line 54b is in registry both with outlet16 of the sample preparation device and inlet 114, and flow line 54c isin registry with outlet 119 of assay structure 112 of the analyticaldevice. As illustrated in FIG. 6A, flow line 54a is in fluidcommunication with appliance inlet port 56, whereas flow line 54c is influid communication with appliance outlet 57. The appliance typicallyincludes an impellent, such as pump 58, for forcing sample fluid throughthe analytical system. After applying to inlet port 56 of appliance 50,a particle-containing fluid test sample, e.g., whole blood, the serumphase of which is suspected to contain an analyte of interest, pump 58is actuated to force the sample through separator 18, providing samplefluid, e.g., serum, of substantially reduced particle content. Thesubstantially particle-free sample fluid is transferred from device 10via flow line 54B to assay structure 112 for testing, e.g., immunoassay.

The binding of analyte, per se, or analyte reaction products to abinding substance in the reaction/detection region of the analyticaldevices can be detected by any number of methods, including monitoringthe pressure or electrical conductivity of sample fluids in thedevice(s), as disclosed in the above-referenced related applications(see, for example, U.S. Ser. No. 877,702 now abandoned), or by opticaldetection through a transparent cover, either visually or by machine.For example, reaction of an analyte with a binding substance in thereaction region 117 of analytical device 112 illustrated in FIG. 6A canbe detected by monitoring the pressure of the sample fluids in certainregions of the mesoscale flow passages. This is accomplished in theanalytical system-appliance combination of FIG. 6A by means of twopressure detectors 59a and 59b for detecting flow pressure of fluidsentering and exiting the devices through ports 14 and 119, respectively.When, during the performance of an assay, particles agglomerate ormolecules chemically interact to form a network causing restricted flowor an increase in the viscosity of the sample liquid passing through thereaction/detection region, such changes can be detected as a pressurechange which is indicative of a positive result. Mesoscale pressuresensors, and other electrical or electromechanical sensors can bedirectly fabricated on a silicon substrate and can be mass-producedaccording to well established techniques. Angell et al., ScientificAmerican, 248: 44-55 (1983).

Other embodiments of appliances may be fabricated for use in carryingout different assay protocols with different devices in accordance withthe present invention. One such embodiment is depicted in FIG. 6B, whichillustrates a cross sectional view of an analytical system, comprisinganalyte device 110' stacked upon a sample preparation device 10',disposed in nesting site 72 provided in appliance 70. Aparticle-containing test sample fluid is applied to appliance sampleinlet 74, whereupon an impellent, such as pump 75, causes the samplefluid to pass through device 10, providing a sample fluid ofsubstantially reduced particle content for analysis in analytical device110'. The cover 116' of analytical device 110' has an aperture 114' opento the atmosphere to vent the system. Placement of the analytical device110' on the top of the stack allows optical detection through atransparent portion of cover 116'.

A separate view of an analytical system, comprising a sample preparationchip and an analytical device for polynucleotide amplification, incombination with an appliance of the type described above is provided inFIG. 7. The cross-sectional view of the analytical system in FIG. 7shows appliance 90 having a nesting site occupied by sample preparationdevice 10 and the polynucleotide amplification/assay structure 122. Thedischarge section 28 of flow channel 24b in sample preparation device 10is in fluid communication, through flow line 92 with the inlet port 124of polynucleotide amplification/assay structure 122. Flow line 93 is inregistry with outlet 129 of the analytical device and in fluidcommunication with appliance outlet 94.

The polynucleotide sample, after release from the cell componentseparated from the sample fluid in sample preparation device 10, e.g.,by contacting with the suitable lysing means as described above, isintroduced into amplification region 127. Reagents required foramplification are also added to amplification region 127 through inlet126, as shown in FIG. 5. An impellent, such as a pump (not shown), isused to deliver the polynucleotide sample through flow line 92 toamplification region 127.

Amplification reagents may be similarly delivered to amplificationregion 127 through a different flow line provided in the appliance or inthe analytical device (not shown). The product of the polynucleotideamplification reaction may be transferred to region 128 for detection inthe manner previously described. The resultant product may be recovered,if desired, through appliance outlet 94.

Pressure differentials along the path of flow of the test sample fluidthrough devices 10 and 122 may be measured using pressure sensor 96 inconjunction with a pressure sensor (not shown) deployed in the applianceor the device to measure pressure at a point upstream of dischargesection 28 of device 10.

Appliance 90 may include a heating/cooling element 95 for controllingthe temperature within the polynucleotide amplification region, e.g., anelectrical heating element and/or a refrigeration element. An electricalheating element (not shown) may alternatively be integrated into thesubstrate of analytical device 122, with electrical elements for powermated to matching electrical contacts in the appliance below theamplification region 127. Alternatively, the appliance may include aninternal or external heating means, such as a laser or other source ofelectromagnetic energy (not shown) disposed adjacent amplificationregion 127 of polynucleotide amplification/assay structure 122. Amicroprocessor in appliance 90 may be used to regulate the heatingelement in order to provide a temperature cycle in the polynucleotideamplification region between a temperature suitable for dehybridization,e.g., 94° C., and temperatures suitable for annealing andpolymerization, e.g., 65° C. A thermocouple may also be provided in thesubstrate surrounding amplification region 127 in electrical contactwith the appliance to allow microprocessor or other electroniccontroller to detect and maintain the temperature cycles in the reactionchamber. A cooling element, such as a miniature thermoelectric heat pump(Materials Electronic Products Corp., Trenton, N.J.), may also beincluded in the appliance for adjusting the temperature of theamplification chamber. In another embodiment, the temperature of thepolynucleotide amplification chamber can be regulated by a timed laserpulse directed at the reaction chamber through glass cover 109, so as toallow sequential heating and cooling of the sample to the requiredtemperatures for the amplification cycle. The thermal properties ofsilicon enable a rapid heating and cooling cycle.

In all of the embodiments of the invention depicted in FIGS. 4, 6A, 6Band 7, the pump may be subject to control by a microprocessor in theappliance. Also, the devices illustrated in the last-mentioned figuresmay be retained securely engaged in the nesting site of the appliance,or in contact with one another, as the case may be, in various waysincluding, by way of example, a clamp (not shown) mounted on theappliance, binding of the confronting device surfaces to one another,e.g., by adhesive, or by appropriate dimensioning the devices relativeto the nesting sites to frictionally retain the devices therein.

A biological assay device which may be used in combination with thesample preparation device of the invention is shown in FIG. 8A. Thedevice 130 was fabricated on a substrate 131 having mesoscale flowchannels 132a, 132b with entry ports 133 microfabricated on oppositeends of the channels and a central mesoscale mixing/capture/detectionchamber 135. As depicted in FIG. 8A, the cross-sectional dimension ofchamber 135 is relatively larger than that of channel 132a, 132b.

A capture reagent, such as a substance that binds specifically to theanalyte of interest, may be immobilized, either on a stationary ormobile support, in chamber 135. When a mobile support, e.g. polymerparticles, is used, the particle size should be selected so as to berelatively larger than the cross-sectional dimension of flow channel132a, 132b in order that the immobilized reagent is confined to chamber135. A reagent immobilized on a particulate solid support in this mannercan conveniently be charged to chamber 135 via inlet port 137.

A device of the type just described can be used to carry out variousimmunoassay reactions. For example, a non-competitive, immunometricassay for the determination of carcinoembryonic antigen (CEA) may becarried out by filling chamber 135 with monoclonal anti-CEA antibodiesimmobilized on a particulate support, such as plastic beads. The testsample to be analyzed for CEA is then added to fill chamber 135 andexpel any fluid introduced with the immobilized reagent. The contents ofchamber 135 are thereafter incubated for a time sufficient to effectantigen-antibody binding. Subsequently, an antibody enzyme conjugate,e.g. monoclonal anti-CEA antibody-horseradish peroxidase is added to thechamber and the contents are again incubated. A solution of achromogenic substrate is then added to chamber 135 which serves to washthe immobilized reagent, expelling unbound conjugate. Sufficientsubstrate is retained in the chamber to react with any peroxidase labelbound to the immobilized reagent. The rate of generation of chromophoreis directly proportional to the concentration of CEA in the sample.

Device 130 may also be used to perform a competitive assay for thedetermination of thyroxine in a test sample. In carrying out thisformat, chamber 135 is filled with an immobilized reagent comprisinganti-thyroxine antibodies bound to the surface of plastic beads. Thetest sample to be analyzed for thyroxine is premixed with athyroxine-peroxidase conjugate and added to the chamber, thus fillingthe chamber and expelling any fluid introduced with the immobilizedreagent. The contents of the chamber are then incubated for a timesufficient to effect antigen-antibody binding. A buffer may optionallybe passed through chamber 135 to wash the immobilized reagent. Achromogenic substrate is thereafter added to the chamber, washing theimmobilized reagent and expelling any unbound reagents. Sufficientsubstrate is retained in chamber 135 to react with any peroxidase labelbound to the immobilized reagent. Generation of chromophore is inverselyproportional to the concentration of thyroxine in the test sample.

Although the assay structure of FIG. 8A is configured to confine theimmobilized reagent in channel 135, the design is such that fluid can bepumped over and through the immobilized reagent for washing purposes.

It should be understood that the last-mentioned two examples are merelyrepresentative, as the device of FIG. 8A, as well as the other devicesdescribed herein may be used to implement a variety of other assayformats.

FIG. 8B shows analytical device 140 microfabricated on a substrate 141and having an inlet port 143 in fluid communication with a chamber 145for analyte capture, e.g., by immunocapture. This device is adapted forcarrying out enzyme immunoassay. To that end, the device includes aseparate chamber 147 containing a binding agent to capture andconcentrate the chromophore produced by the action of the enzyme labelon a suitable substrate. For example, a protein analyte may bedetermined using a "sandwich" assay technique, in which the analyte iscaptured in chamber 145 by an antibody immobilized therein which bindsspecifically to the analyte. The captured analyte is labelled with anenzyme-antibody conjugate composed of alkaline phosphatase, for example,and an antibody that specifically binds the protein analyte. Fluoresceinphosphate is introduced into chamber 145 as a chromogenic substrate forthe enzyme label. Alkaline phosphatase acts on the substrate to generatefluorescein which is captured by an anti-fluorescein antibodyimmobilized in chamber 147. A hydrophobic environment created in chamber147, e.g., by virtue of material adhered to the walls of the structure,the capture agent or a component of the reaction mixture, e.g., asurfactant or micelle-forming agent, will improve the fluorescent signalfrom the bound fluorescein. Detection of the chromophore may be carriedout in chamber 147 or the chromophore may be removed from the devicethrough outlet 149 for detection in a separate apparatus. Othersubstrates could be selected for use in carrying out this determination,such as 4-nitrophenol phosphate or 4-methylumbelliferone phosphate, withappropriate binding agents used to capture the dephosphorylated product.

A diagrammatic representation of another embodiment of a biologicalassay device that may be used in the practice of the present inventionis shown in FIG. 9. The substrate 151 of device 150 is microfabricatedwith ports 152a-e, flow channels 154a-g, reaction chambers 156a and 156band a capture/detection chamber 158. The reaction chambers 156a and 156beach comprise a tortuous mesoscale flow channel. The path length of thetortuous channel may be designed to permit the timed mixing and additionof sample reagent(s). Devices of this type may be utilized incombination with an appliance having ports mated to ports in the device,which appliance is capable of delivering and receiving fluids throughthe flow system of the device and, optionally, capable of opticallydetecting a positive or quantitative result in chamber 158. In oneapplication of the device, the cholesterol content of a sample may bedetermined. Cholesterol esterase is applied via inlet port 152a andbuffer and sample are added via inlet ports 152b and 152c, respectively.The mixture then flows through channel 154d to the tortuousmixing/reaction chamber 156a. The time of mixing and reaction may bepredetermined by microfabricating the tortuous channel to theappropriate length and controlling the flow rates. Cholesterol oxidaseis added via port 152d and flows through channel 154g to the tortuouschannel 156b where the timed mixing and reaction of the cholesteroloxidase with the fluid from channel 156a occurs. Heating means likethose described above, may be provided to maintain the device at 37° C.,or higher. A chromogenic substance is introduced at 154e through a flowchannel (not shown) for detection. Positive or quantitative results canbe detected optically by observing the detection chamber 158, e.g.,through an optical window disposed over the chamber. The detectionchamber 158 may be provided with an immobilized binding moiety capableof capturing the product of the enzyme reaction, thus facilitatingdetection. This device may be applied to a range of clinical enzymaticand other reactions.

According to an alternative embodiment shown in FIG. 9B, capture of afluorescently labelled analyte may occur in chamber 158a, which containsan analyte-specific binding agent that binds releasably to the analyte.Released fluorescently labelled analyte is captured for detection inchamber 158b.

In another embodiment illustrated in FIG. 9C, flow channel 154f may beconstricted, such that the flow path is of smaller cross-sectional areathan channel 154e, thereby restricting flow of test fluid through thedevice. As depicted in FIG. 9C, channel 154f is constructed in a patternof parallel flow channels, with reduced dimensions at each channeldivision, providing sequentially narrower flow passages. This device maybe utilized in performing various agglutination assays, the occurrenceof particle-induced or complex-induced agglutination being detected onthe basis of restricted flow of the sample through the branched portion159 of flow channel 154f.

FIG. 10A is a diagrammatic representation of a mesoscale analyticaldevice 170 design for carrying out various binding assay protocols. Thedevice enables determination of a range of analytes on the basis ofmicrovolumes of sample and small, measured amounts of reagents, withlabelled product being detected within the device, so that all sample,unreacted reagent and reaction products remain confined in the devicefor subsequent disposal.

The device may be used in combination with an appliance (not shown) ofthe general type described above with reference to FIG. 6A. Such adevice has a nesting site for holding the device, flow lines andassociated pumps and valves for delivering sample, reagents, washsolutions and the like to the device. The appliance may also include atemperature control and sensing means, pressure sensors and/orelectrical connections to facilitate analyte detection, opticaldetection means, signal amplification and quantitation means, all asdescribed herein. The combination may also include overall systemsequence and control elements, quantitated information display andrecording means via a microprocessor in the appliance, for example, orby interfacing with an external computer.

The device is microfabricated as previously described with the flowpassages configured to provide a total capacity in the range of 0.01-100μL, preferably from about 0.5 to about 50 μL.

In use, a microvolume of test sample fluid is introduced at port 171.The test sample fluid may be pre-filtered, e.g., by passage through thesample preparation device of the invention, before introduction at port171. Alternatively, the sample fluid may be filtered after introductioninto device 170. Internal filtration may be beneficially achieved by across-flow filtration technique. As shown in FIG. 10B, flow passage 172,through which sample fluid initially passes upon introduction at inlet171, is divided into two side-by-side V-shaped channels 172a and 172b,separated by a longitudinal barrier 173, which is preferably formed fromthe substrate material (but may be a part of, and suspended from thecover plate or sheet). Barrier 173, together with the cover of thedevice, defines at least one passageway 174, as illustrated in FIG. 10C,which allows fluid flow therethrough, but is of sufficiently smalldimension to prevent the passage of particulate components, e.g., cells,of a fluid sample. Barrier 173 is positioned such that inlet 171 feedssample fluid directly into flow path 172a and indirectly into flow path172b, the fluid passing into flow path 172b having a substantiallyreduced particle content, as compared with previously unfiltered sampleentering inlet 171.

Flow passage 172 may be fabricated with walls that diverge from arelatively small cross-sectional dimension to a relatively largercross-sectional dimension in the downstream direction from the inlet, orwith walls that converge from a relatively large cross-sectionaldimension to a relatively smaller cross-section dimension in thedownstream direction from the inlet, with barrier 173 being disposedgenerally parallel to at least one of the passage walls. Such designgives rise to non-linear flow of the sample fluid which aids indislodging particles from passageway 174.

If the test sample fluid is filtered externally to device 170, theabove-described internal filter may be omitted. Alternatively, a samplefluid that has been externally filtered can be entered directly into thedevice via port 175, thus bypassing flow passage 172. A buffer may alsobe introduced through port 175 for the preparation of diluted samplefluid, if desired. Excess buffer may be collected in outlet 176.

Particulate matter trapped in flow path 172a is conveyed to outlet 176,as illustrated in FIG. 10B.

Filtrate from flow path 172b next passes into flow passage 177 which isappropriately dimensioned to function as a metering chamber, providing apre-determined sample volume for analysis. The pre-determined samplevolume will ordinarily be on the order of about 1 μL. A scale 178 may beprovided on device 170, e.g., by etching, to aid in the metering ofdesired amounts of sample fluid into the device for analysis. Byenabling the introduction of prescribed sample volumes into device 170,flow passage 177 also permits quantitation of the analyte.

A suitable impellent (not shown) incorporated in device 170, or in anappliance designed for use in conjunction with such device, can beemployed for transferring the metered sample fluid to flow passage 179,which is optionally provided for mixing the sample fluid with theprimary reagent used in performing the binding assay. The inclusion ofsuch a mixing chamber in device 170 is beneficial for achieving morerapid and complete reaction between analyte and primary reagents.

Suitable impellents for transferring sample fluid, reagents, buffers andthe like through the flow system of device 170 includes various pumps,such as micromachined pumps, diaphram pumps, syringe pumps, volumeocclusion pumps, as well as endosmotic induced flow, flow induced byelectrochemical evolution of gases and other pumping means known tothose skilled in the art.

The primary reagents may be delivered directly to flow passage 179 inthe device through inlet 180. The primary reagents are caused to mixwith the metered sample fluid upon entering flow passage 179, which maybe sequential or essentially simultaneous. Excess primary reagents maypass out of the flow system through outlet 181.

The source of primary reagent may be an internal storage chamber whichcan optionally be provided in device 170. Alternatively, the primaryreagents can be delivered to the device from a reservoir in an appliancewith which the assay device is used, such as the appliance describedwith reference to FIG. 6A, above, or from some other source external tothe device. The primary reagents can be stored as liquid solutions, gelsor neat, such as in dried or lyophilized form, or in any otherconvenient form. For example, the primary reagent can be lyophilized inplace in flow passage 179, in which case the test sample fluid or asuitable solvent introduced, for example, through inlet 180 can be usedto dissolve the primary reagents. Alternatively, the test sample or asolvent may be directed by liquid transfer means, as noted above, fromflow channel 179 to a storage chamber (not shown) outside the flowsystem illustrated in FIG. 10 to dissolve the primary reagents. Inaddition, heating or agitation means (not shown) may be provided in thestorage chamber to aid in dissolving the primary reagents storedtherein.

The primary reaction mixture, comprising the sample fluid and dissolvedprimary reagents can also be reacted in flow channel 179, which mayinclude structural elements, as previously described, to promoteturbulent flow. Agitation or other means may be provided to ensureadequate mixing of the primary reaction mixture. The primary reactionmixture is caused to remain in flow channel 179 for a time sufficientfor the desired reaction to proceed to completion.

Means for regulating the temperature in flow channel 179, such as thatpreviously described with reference to FIG. 7, may optionally beutilized to enhance the primary reaction conditions. Means for sensingthe temperature in flow passage 179 may also be provided, if desired.The temperature sensing means may be operatively connected to amicroprocessor or similar device which controls the overall function ofthe system so as to correlate the sensed temperature with the residencetime of the primary reaction mixture in flow passage 179.

Upon completion of reaction, all or part of the primary reaction mixturecan be transferred, e.g., by the above-described pumps or otherimpellents, to capture region 182 and detection region 183, in which oneor more original components of the sample fluid or products of theprimary reaction may be monitored and/or detected. Alternatively, theproduct of a secondary reaction, the existence or concentration of whichis correlatable to the existence or concentration of the analyte ofinterest in the sample fluid, can be employed for analyte determination.

The detection techniques utilized in connection with device 170 arethose customarily used in performing binding assays. Briefly, theseinclude chemical tests, such as may be carried out by addition of testreagents; spectroscopy, for example, to detect changes in properties ofthe analyte caused by chemical changes during the primary reaction, suchas shifts in absorbance, wave lengths, changes in fluorescencepolarization, changes in fluorescence stokes shifts, and the like;agglutination, as measured by microscope, image analysis or similarprocedures; and measuring electrochemical performance of the reactedprimary reaction mixture, such as specific measurement by amperometricand/or potentiometric/voltametric techniques.

With regard to carrying out a secondary reaction for analytedetermination, a capture region, defined by flow passage 182, isprovided into which all or part of the reacted primary reaction mixtureis transferred by liquid transfer means of the type previouslydescribed, and in which one or more components of the products in theprimary reaction mixture may be captured by binding to a surface andsubsequently detected and/or quantitated. Capture reagent may beimmobilized on the walls of flow passage 182 or on the surface ofparticles or beads present in flow passage 182, or both.

An inlet or fill hole 184 may be provided to pre-fill flow passage 182with solid phase capture reagent comprising plastic, latex, silica orother suitable support material, including magnetic components, capableof combining specifically to the products of the primary reactionmixture. The particulate capture reagent can be charged to flow passage182 either as a wet slurry, which may subsequently be dried orlyophilized, or in dry form. In either case, the filling of flow passage182 can optionally be assisted by vibration or other means. The mobilesolid phase of the capture reagent comprises particles or beads havingdiameters from tens of nanometers to tens of microns, with a surfacecoating of avidin, strepavidin or other substance to which biotinylatedor otherwise conjugated antibodies will specifically bind.

Flow passage 182 may be fabricated with flow restricting structuralelements 189a, 189b or other means to confine the capture reagent withinflow passage 182 while allowing passage of fluids therethrough. Theparticulate capture reagent may also be confined within flow passage 182in the manner previously described with reference to FIG. 8A.

The primary reaction mixture is caused to remain in flow passage 182 fora time sufficient for reaction with the capture reagent to proceed to aknown extent, preferably essentially to completion. Means for regulatingand sensing the temperature in flow passage 182 may optionally beprovided as noted above with reference to flow passage 179.

The captured product of the primary reaction mixture is preferablywashed before proceeding with the secondary reaction.

The reagent solution for the secondary reaction may be delivereddirectly to device 170 via inlet 185. Excess secondary reagent may beremoved from the flow system through outlet 186 or 187. Alternatively,the reagent for the secondary reaction may be kept prior to dissolutionand use in a storage chamber in device 170, or in an appliance used inconjunction with the device, or in some other convenient source externalto the device. One or more flow lines appropriately mated with flowpassages in device 170 and operatively connected to an impellent mayoptionally be provided to transfer solvent from an input port to theabove-mentioned secondary storage chamber where stored reagents aredissolved to form the secondary reaction solution.

The reagent for the secondary reaction may include an enzyme substratespecific to an enzyme conjugated to the captured primary reactionproduct, as well as substances which, when dissolved in the secondaryreaction solution, assist in washing of the bound primary reactionproduct.

The secondary reaction preferably occurs in flow passage 182, whereinthe secondary reaction solution reacts with captured primary reactionproducts. The product of the secondary reaction may be a substanceselected from the group of molecules or ions directly or indirectlydetectable based on light absorbance, fluorescence, phosphorescenceproperties; molecules or ions detectable by their radioactiveproperties; or molecules or ions detectable by their nuclear magneticresonance or paramagnetic properties. The product of the secondaryreaction may be amplified, according to procedures known in the art toenhance the detection thereof. For example, an enzyme amplificationreaction may be employed, which releases a florophore generated from anon-fluorescent precursor in the secondary reaction solution.

After the secondary reaction is complete, the resultant product may bedetected and quantitated either within flow passage 182 or subsequentlyin detection region 183, or in a detector external to device 170.

The preferred cross-sectional dimensions of flow passages 177 and 183,transverse to the path of flow of sample fluid, are about 100 μm wideand 70 μm deep, whereas the preferred cross-sectional dimensions of flowpassages 179 and 182, transverse to the path of flow of sample fluid,are about 400 μm wide and 70 μm deep. These dimensions are within themesoscale range, as set forth above.

Various binding assay protocols can be implemented in device 170including immunometric (sandwich) assays as well as competitiveimmunoassays, employing both polyclonal and monoclonal antibodies forpurposes of capture and detection of analyte. One form of detectionantibody comprises a conjugated label wherein the label is florophoredetectable as a bound moiety after capture on a solid phase. Anotherform of detection antibody comprises a conjugated label wherein thelabel is florophore detected after release from the captured primaryreaction product. Another form of detection antibody comprises aconjugated enzyme moiety such as horseradish peroxidase or alkalinephosphatase.

Washing steps may be carried out as appropriate to eliminate potentiallyinterfering substances from device 170.

Excess sample fluid, reagents, wash solutions and the like from thevarious flow passages and structural elements may be combined and routedinto a single waste receptacle of adequate capacity, preferably withindevice 170, such that all sample fluid and reaction products are safelycontained for disposal.

FIG. 11A diagrammatically depicts an analytical device 191 used todetermine the presence of an intracellular polynucleotide in abiological cell-containing fluid sample, and then to perform an assayfor a particular nucleotide sequence. Microfabricated on substrate 192is a mesoscale flow path 194a-c which includes a cell separation chamber196a, a cell lysis chamber 196b, a filter element 197, a polynucleotideamplification chamber comprising sections 198a and 198b, and a detectionregion 199. The mesoscale flow system is also provided with fluidentry/exit ports 193a-d. The device can be used in combination with anappliance, such as that described above with reference to FIG. 6A.

Initially, the valves in the above-mentioned appliance function to closeports 193c and 193d, while ports 193a and 193b are open. A samplecontaining a mixture of cells, e.g., transferred from the samplepreparation device, is directed to the sample inlet port 193a by asuitable impellent, e.g. a pump, (not shown), and flows through themesoscale flow channel 194a to separation chamber 196a. Chamber 196acontains binding moieties immobilized on the wall of the chamber whichselectively bind to a surface molecule on a desired cell type in thesample. Remaining cellular components exit the substrate via port 193b.After binding of the desired cell type in chamber 196a, flow with bufferis continued, to wash and assure isolation of the target cells. Nextport 193b is closed and 193c is opened. Flow is then increasedsufficiently to dislodge the immobilized cells from chamber 196a. Flowis continued, forcing cells through membrane piercing protrusions 195 inchamber 196b, which tear open the cells releasing intracellularmaterial.

Sample flow continues past filter 197, which filters off large cellularmembrane components and other debris, with the filtrate passing tomesoscale PCR chamber section 198a, which is connected to PCR chambersection 198b by flow channel 194b. Taq polymerase, primers and otherreagents required for the PCR assay next are added to section 198bthrough port 193c from a source thereof (not shown), permitting mixingof the intracellular soluble components from the separated subpopulationof cells and the PCR reagents. With the ports closed (to ensure that thereaction mixture does not evaporate, or otherwise becomes lost from thedevice), an impellent, e.g. a pump, (not shown), applies a motive forceto port 193b to cycle the PCR sample and reagents through flow channel194b between sections 198a and 198b, set at 94° C. and 65° C.,respectively, to implement plural polynucleotide melting andpolymerization cycles, allowing the amplification of the polynucleotideof interest. Before the next process step, port 193c is closed and port193d is opened. The same impellent force is then used to direct theamplified polynucleotide isolated from the cell population to adetection region 199 in the form of a pattern of flow channels like thatdescribed above with reference to FIG. 9C. Flow reduction in therestricted region serves as a positive indicator of the presence ofamplified polynucleotide product and may be detected optically through aglass cover disposed over the detection region 199. Alternatively, theamplified polynucleotide product may be detected directly in thereaction chamber, using commercially available reagents developed forsuch purpose, such as the "Taq Man®" reagents, available from PerkinElmer Corporation. The amplified polynucleotide may also be detectedoutside the device using various methods known in the art, such aselectrophoresis in agarose gel in the presence of ethidium bromide.

Another embodiment of an analytical device which is useful in thepractice of this invention is illustrated in FIG. 11B. The device 210comprises a substrate 214 microfabricated with a mesoscalepolynucleotide amplification chamber 222A. The device 210 can be used incombination with an appliance like appliance 90 shown in FIG. 7. Theappliance is provided with flow paths mated to ports 216A, 216B, 216Cand 216D in device 210. The appliance may also include valves that allowthe ports 216A, 216B, 216C and 216D to be mechanically opened andclosed. In one embodiment, the flow system of the devices may bemaintained at a hydraulically full volume, and valves in the appliance,or alternatively, in the devices themselves, may be utilized to directfluid flow. Chamber 222A is heated and cooled to temperaturesappropriate to provide a dehybridization temperature, and annealing andpolymerization temperatures, as required for PCR. Temperature of thereaction region can be controlled as previously described with referenceto FIG. 7.

The flow system illustrated in FIG. 11B includes filter elements 224, ofthe general type described herein, to remove from the sample fluidfilterable components having a tendency to interfere with the analysis.

In operation, a sample containing polymerase enzyme and other reagentsrequired for PCR is delivered through inlet port 216A to reactionchamber 222A. With the ports closed, a heating element is then utilizedto thermally cycle the reaction chamber between a temperature suitablefor dehybridization and temperatures suitable for annealing andpolymerization. When the PCR reaction cycle is terminated, ports 216Band 216D are opened, driving the contents of chamber 222A to detectionregion 222B, which region contains a polynucleotide probe, e.g.,immobilized upon beads 292. A positive assay for the polynucleotide isindicated by agglutination of the beads in the detection region.

Although polynucleotide amplification has been described herein withparticular reference to PCR, it will be appreciated by those skilled inthe art that the devices and systems of the present invention may beutilized equally effectively for a variety of other polynucleotideamplification reactions. Such additional reactions may be thermallydependent, such as the polymerase chain reaction, or they may be carriedout at a single temperature (e.g., nucleic acid sequenced-basedamplification (NASBA)). Moreover, such reactions may employ a widevariety of amplification reagents and enzymes, including DNA ligase, T7RNA polymerase and/or reverse transcriptase, among others. Additionally,denaturation of polynucleotides can be accomplished by known chemical orphysical methods, alone or combined with temperature change.Polynucleotide amplification reactions that may be practiced in thedevice of the invention include, but are not limited to: (1) targetpolynucleotide amplification methods such as self-sustained sequencereplication (3SR) and strand-displacement amplification (SDA); (2)methods based on amplification of a signal attached to the targetpolynucleotide, such as "branched chain" DNA amplification (ChironCorp., Emeryville, Calif.); (3) methods based on amplification or probeDNA, such as ligase chain reaction (LCR) and QB replicase amplification(QBR); (4) transcription-based methods, such ligation activatedtranscription (NASBA); and (5) various other amplification methods, suchas repair chain reaction (RCR) and cycling probe reaction (CPR) (for asummary of these methods and their commercial sources, see pp. 2-7 ofThe Genesis Resort, DX, Vol. 3, No. 4, February 1994; Genesis Group,Montclair, N.J.).

The sample preparation device of the invention may be used inconjunction with Mesoscale Polynucleotide Amplification Devices, whichis the subject matter of U.S. Ser. No. 08/308,199, now U.S. Pat. No.5,498,392. The entire disclosure of the last-mentioned application isincorporated by reference herein.

Briefly, the last-mentioned patent application relates to mesoscaledevices for amplification of a preselected polynucleotide in a samplefluid. The devices are provided with a substrate microfabricated toinclude a polynucleotide amplification reaction chamber having at leastone cross-sectional dimension of about 0.1 to 1000 μm. The device alsoincludes at least one port in fluid communication with the reactionchamber, for introducing a sample to the chamber, for venting thechamber when necessary, and, optionally, for removing products or wastematerial from the device. The reaction chamber may be provided withreagents required for amplification of a preselected polynucleotide. Thedevice also may include means for thermally regulating the contents ofthe reaction chamber, to amplify a preselected polynucleotide.Preferably, the reaction chamber is fabricated with a high surface tovolume ratio, to facilitate thermal regulation. The amplificationreaction chamber also may contain a composition which diminishesinhibition of the amplification reaction by material comprising a wallof the reaction chamber, when such treatment is required.

Appliances 30, 50, 70 and 90, as shown in FIGS. 4, 6A, 6B and 7,respectively, may also be utilized to deliver metered amounts of sample,reagent buffer and the like, as well as to implement the timed additionof sample or other fluids to the devices in connection with theperformance of prescribed analytical protocols.

In those cases where a microprocessor is included in the appliance itmay be used to assist in the collection of data for one or a series ofanalyses.

Although analyte determination has been described above with particularreference to whole blood as the sample fluid, the analyte of interestmay be present in test samples or specimens of varying origin, includingother biological fluids such as whole blood containing anti-coagulants,dilute whole blood, lysed whole blood, whole blood containing assayreagents, serum, plasma, urine, sperm, cerebrospinal fluid, amnioticfluid, lavage fluids, tissue extracts, cell suspensions and any othersample fluid that can be beneficially analyzed using the device andsystems described herein.

FIGS. 12A-D illustrate various additional embodiments ofmicrofabricated, restricted flow separators which may be disposed in theflow passages of the devices described herein. The separator in FIG. 12Ais in the form of a plurality of partitions 251, projecting fromopposite surfaces 252a, 252b of channel 253, so as to define a series ofpassageways 254a, 254b, which are aligned longitudinally along thechannel. One or more intermediate partitions 255, projecting from thebottom of channel 250 may be disposed adjacent the downstream-facingportion of one or more of partitions 251, to stand as barriers orbaffles within the flow path provided by aligned passageways 253.

Sample fluid passing through the relatively narrow passageways 254a,254b at relatively high speed will tend to disperse into the spacebetween consecutive partitions, while reducing in speed and moving intothe dead volume corners of such space. When sample fluid then passesinto the next successive inter-partition space, particulate matter maybe relatively retained in the dead volume. Thus, for each passage into asubsequent inter-partition space, particulate matter is progressivelyretained and sample fluid becomes gradually more purified as it flowsdownstream through the partitions. With a sufficient number ofpartitions in series, progressive reduction in particle concentrationwould be enabled, the efficiency of which could be predetermined.Baffles 255 would assist in directing the sample fluid into the deadvolume region.

In FIG. 12C, there is shown a weir-type separator structure formed bybarriers 257 projecting up from the bottom 258 of channel 250.

The separator structure shown in FIGS. 12C and 12D takes advantage ofthe propensity of particles to fall under the influence of gravity. Thismay be particularly useful in the analysis of whole blood, by promotingthe sedimentation of erythrocytes. The sample fluid passes at high speedover barrier 257, then immediately slows. Any particulate matter fallingtowards the floor of channel 250 will experience a lower supportingvelocity and a diminished opportunity of being swirled up over the nextsucceeding barrier. Passage of sample fluid over a series of suchbarriers may progressively reduce particulate concentration and producegradually more purified sample fluid. One or more lips 259 suspendedfrom cover plate 260 assists in downwardly directing the sample fluid.

The following examples are provided to describe the invention in furtherdetail. These examples are intended to illustrate and not to limit theinvention.

EXAMPLE 1

A plastic-silicon composite assay device was fabricated by attaching aplastic (3M transparency sheet) cover over a silicon substrate 131,microfabricated with flow channels 132a, 132b having entry ports 133 onopposite sides of the channel and a central reaction/detection chamber135, as shown schematically in FIG. 8A. A dilution of anti-A (in 0.05 Msodium bicarbonate pH 9.6) and a 1:10 dilution of Type A blood in salinewere introduced via syringe using a holder into the entry ports 133 onopposite ends of the channel 132a, 132b. The solutions mixed together inthe central chamber 135 and agglutination was observed through theplastic cover by light microscopy. The results are summarized in thefollowing table.

    ______________________________________                            AGGLUTINATION IN    ANTI-A        DILUTION  CHANNEL    ______________________________________    Gamma Kit     1:20      +    Gamma Murine Mono                  1:20      +    Gamma Human Dilution                  1:5       +    Immucor Affinity pure                   1:100    +    Immucor Ascites                   1:100    +    ______________________________________

EXAMPLE 2

A solution of mouse IgG (50 μg/mL in 0.05 M sodium bicarbonate pH 9.6)(SIGMA Cat. No. 1-5381) and a 1:20 dilution of goat anti-mouse IgG(H&L)--fluorescence carboxylate beads (Polysciences, Inc.) in PBS bufferwere introduced via syringe using a holder into the entry ports onopposite ends of channels 132a, 132b in another assay device prepared asdescribed in Example 1. The solutions were mixed together in thereaction/detection chamber 135 and agglutination was observed throughthe transparent plastic cover by light microscopy.

While certain embodiments of the present invention have been describedand/or exemplified above, various other embodiments will be apparent tothose skilled in the art from the foregoing description. The presentinvention is, therefore, not limited to the particular embodimentsdescribed and/or exemplified, but is capable of considerable variationand modification without departure from the scope of the appendedclaims.

What is claimed is:
 1. A method of separating a target subpopulation ofcells in a cell-containing sample fluid comprising the steps of:a)providing a solid substrate microfabricated to define a mesoscale samplefluid flow passage in fluid communication with a mesoscale chamber, saidchamber and said flow passage being dissimilar in their cross-sectionaldimensions, said chamber having a wall portion defining an enclosure inwhich is disposed an immobilized binding protein specific for a cellmembrane-bound protein characteristic of said target population; b)passing a cell-containing sample fluid through said enclosure underconditions to permit capture of members of the cell target subpopulationby reversible cell surface protein-immobilized protein binding, whilepermitting other cells to pass therethrough; and c) controlling theenvironment in said enclosure to effect release of said targetsubpopulation of cells.
 2. A method of separating a first material froma fluid containing a mixture of said first material and other material,comprising:providing a mesoscale sample preparation device whichcomprises a solid substrate microfabricated to define a mesoscale flowchannel in fluid communication with a mesoscale chamber, said chamberand said flow passage being dissimilar in their cross-sectionaldimensions, said chamber having a wall portion defining an enclosure inwhich is disposed an immbolized specific binding substance whichspecifically binds said first material; passing said fluid containingsaid mixture through said enclosure under conditions to permit captureof said first material, while permitting said other material to passtherethrough; and controlling an environment in said enclosure to effectrelease of said first material from said specific binding substance. 3.The method of claim 2, wherein the first material is an antigen and thespecific binding substance comprises an antibody which specificallybinds said antigen.
 4. The method of claim 3, wherein the first materialis a product of an enzyme reaction.
 5. The method of claim 2, furthercomprising the step of detecting the first material released in saidcontrolling step.
 6. The method of claim 2, wherein said first materialis labeled with a group selected from a fluorescent labeling group and acolorimetric labeling group.
 7. The method of claim 2, wherein saidenclosure comprises a transparent cover.